3.2. Markers of CKD
As previously mentioned, chronic kidney disease (CKD) is a polyetiological disease. The primary issue with CKD is that the inflammatory response and impaired renal function are accompanied by processes that partially sustain them. CKD is linked to various osmolytes and uremic solutes, which are associated with biochemical pathways in the body.
Uremic solutes are toxic compounds. They are associated with uremia and subsequent changes in organ function. Robert D. Mair and colleagues performed urine metabolomic analysis by using LC-MS to study the clearance of uremic solutes [55]. However, this approach required urine and plasma samples to calculate the fractional clearance formula. Specifically, in patients with advanced disease, the most significant changes in clearance compared with control samples were observed for p-cresol sulfate, indoxyl sulfate, hippurate, and phenylacetylglutamine.
This is consistent with the results of another study in which elevated indoxyl sulfate levels in urine were also correlated with elevated serotonin sulfate levels. The authors suggested that both markers could be considered significant signals for the early diagnosis of CKD [56]. However, it is uncertain whether a single molecule can definitively resolve the issue of delayed CKD diagnosis. Targeting analysis of compounds related to indoxyl sulfate metabolism would be valuable to confirm these findings.
Osmolytes are the compounds involved in cellular osmotic regulation, which alters CKD. Ryan B. Gil and colleagues confirmed the altered concentration of uremic toxins (e.g., hippuric acid, indoxyl sulfate, and p-cresol sulfate) and included osmolytes in the developed diagnostic panel [14]. Two of the most important protective compounds from the osmotic stress effect are betaine and myo-inositol. These molecules showed the best prognostic value, also agreeing with eGFR. The confirming transcriptomic analysis accompanied these results on a murine CKD model.
In addition to osmotic stress, kidney tissue is also affected by oxidative stress during the development of CKD. Markers of this process were observed in the Balkan Endemic Nephropathy (BEN) study. Additionally, the uremic toxin p-cresol was detected in the urine of a group of patients.
Amino acids (AAs) undergo filtration and reabsorption in the kidney. Altered reabsorption of AAs in the proximal tubule may explain the increased urinary levels and decreased serum levels of AAs in patients with chronic kidney disease (CKD). With progressive renal dysfunction, the urinary levels of AAs increase, indicating impairment of both glomerular and tubular structures, even in the early stages of CKD. In the study by Galavan et al., phenylalanine was also included in the metabolomic profile of CKD [56]. This amino acid has been observed by researchers investigating different subtypes of CKD. This can be explained by the data published this year on phenylalanine hydroxylase (PAH) activity. PAH is commonly found in the kidney, liver, and pancreas.
3.3. Markers for CKD Subtypes
Diabetic kidney disease (DKD) is a highly prevalent form of CKD and is most commonly studied in the context of urinary metabolomics. However, several challenges are involved in formulating the format of a DKD study. Diabetes mellitus is a lifelong endocrine disease, and the manifestation of CKD can be detected at any time and at different rates. As a result, highly sensitive progression markers are essential.
Progression markers of DKD were examined in various experimental models. One approach involved annually evaluating the slope of a clinical parameter such as eGFR
[9][27][32][57], time until the end stage
[57], and time until incident kidney failure with replacement therapy
[32]. An alternative method involved dividing the study cohort into groups based on the stage of DKD before comparing the samples
[18].
These results are consistent with the discovery of 3-hydroxyisobutyrate (3-HIBA) metabolite, which was identified as a catabolic intermediate of BCAA in a study of CRIC participants with diabetes
[32]. Impairment in BCAA catabolism plays a significant role in the pathogenesis of type 2 diabetes and obesity
[58]. In the work by Chasapi et al., 3-methyl-2-oxovalerate, a compound involved in BCAA catabolism, served as a diagnostic element to differentiate hypertensive nephrosclerosis from diabetic nephropathy
[16]. The changes in the TCA cycle and BCAA catabolism could be associated with the decline of mitochondrial functions, angiogenesis, or the development of insulin resistance and ketoacidosis in diabetic patients
[32]. However, in the non-diabetic cohort, both the individual and total levels of BCAAs showed a moderate probability of early-stage cardiovascular, metabolic, and renal disease, with the strongest likelihood being for the latter
[59].
But not only BCAA amino metabolism has been mentioned in recent works. Other amino acid pathways have been included in metabolomic diagnostic panels. Taherkhani et al. underlined the importance of aromatic amino acid metabolism in the early stage of contrast-induced nephropathy
[26][60][61]. However, in the work of J.R. Lucio-Gutiérrez, et al., compounds related to amino acid metabolism and biosynthesis were associated with moderate and severe DKD
[18].
The TCA cycle in mitochondria has adenosine triphosphate (ATP) production function and regulates glucose, fatty acid, and amino acid metabolism
[61][62]. It was shown that deterioration in mitochondrial processes accompanies non-diabetic CKD
[14] and DKD
[9][18][32]. TCA cycle impairment could also be related to regulating inflammation and oxidative stress
[60][61].
Compounds related to fatty acid metabolism are also common in DKD diagnostic panels. Elevated albumin excretion and fatty acid loads filtrated by the glomerulus lead to the reabsorption of a bounded free acid surplus with albumin in the renal tubules
[63]. Other processes connected with fatty acids’ metabolism and mentioned in the literature are kidney function maintenance
[64], podocyte apoptosis by reactive oxygen species (ROS) formation
[65], and keto acid metabolism
[64]. Linoleic acid and γ-linolenic acid uremic concentrations have a dramatic difference in the ADKD group in comparison with the simple DM and NADKD groups
[35].
Another significant feature, as mentioned previously, is the rate at which chronic kidney disease (CKD) develops. Hirakawa et al. designed a marker panel for the rapid decline in DKD, using a combination of non-targeted metabolomics and advanced machine learning analysis
[27]. These methods improved the data scope that could be extracted from patient samples. The urinary threonic acid was found to be a better marker of rapid decline in DKD. The role of threonic acid in pathogenesis is unclear due to the limited metabolomic panels reporting on it
[27]. The study’s main strength is the high number of recruited patients, making the results especially significant. In another smaller cohort, some patients exhibited rapid progression of kidney dysfunction and changes in phospholipid metabolism. This could be due to the involvement of LPC (16:0 and 18:0) in the metabolic shift during DKD progression.
3.4. Markers of Acute Kidney Injury
Acute kidney injury (AKI) is defined as a rapid decline in kidney function. AKI can be viewed as a potential risk factor for the development of CKD and a consequence of CKD. However, AKI and CKD may coexist and interact in a bidirectional fashion. AKI can accelerate the progression of CKD, while CKD can increase one’s predisposition to AKI. This interaction can lead to a cycle in which AKI worsens CKD, increasing the risk of future AKI episodes. Both conditions may have similar symptoms, and diagnosing AKI can be as complex as diagnosing CKD.
Chen Chaoyi et al. found that a diagnostic panel for AKI that included xenobiotic metabolism played a critical role in disease manifestation
[28]. Samples from healthy patients could be differentiated from those with AKI by identifying compounds related to xenobiotic metabolism, such as cytochrome P450 and arginine and proline metabolism
[28].
3.5. Markers of Renal Impairment in Children
Chronic kidney disease (CKD) in children shares the same diagnostic problems but has additional limitations in treatment and transplantation. However, research on pediatric cohorts is limited. In papers describing metabolomic profiles in children, the number of samples is exceptionally small, and population studies are lacking.
Diagnostic metabolomic panels typically include markers associated with the TCA cycle and amino acid and fatty acid metabolism, as noted above. This is also true for studies based on the results of pediatric cohort analyses
[10][19][31][66][67].
Maciozsek et al. performed a metabolomic analysis to investigate significant variations in biochemical pathways in children who had been diagnosed with renal dysplasia
[10]. The results showed a decrease in the levels of acylcarnitines, indoxyl sulfate, xanthine, glutamine, and aconitate, accompanied by increased levels of lactate, dimethylguanosine, and guanidinosuccinate in the patients’ urine. Based on the collected data, the authors concluded that the regulatory processes of glycolysis (the Warburg effect), the ornithine cycle, the tricarboxylic acid cycle, purine metabolism, and fatty acid biosynthesis are disrupted in children with renal dysplasia
[10].
4. Conclusions
CKD is a challenging disease. Because of its specific pathological course, inflammation that starts in one part of the organ gradually spreads to functional kidney tissues. The existing diagnostic methods are insufficient, resulting in a delay in detecting this process. However, there is a promising opportunity for diagnostic method development based on a metabolomic approach. Typically, urinary metabolic studies in CKD patients utilize NMR and tandem MS techniques. However, not every medical center is equipped to perform analysis using expensive MS and NMR analyzers. These instruments’ limited availability and high cost hamper the widespread use of metabolomic screening methods. This poses a challenge in developing and optimizing methods that align with the specific characteristics of the clinical laboratory. In addition, matrix effects and high initial sample dilution must be taken into account when developing urinalysis methods for metabolomics.
The metabolomic approach presents vast potential for advancing personalized medicine related to CKD. However, additional research is needed to address the challenges of cost, availability, validation, and standardization of urine metabolomic analysis to maximize the benefits of metabolomics in the diagnosis and prognosis of CKD.